Immunotherapy involving the priming and expansion of cytotoxic T lymphocytes (CTLs) holds promise for the treatment of cancer and infectious diseases, particularly in humans (Melief et al.,Immunol. Rev. 145:167-177 (1995); Riddell et al., Annu. Rev. Immunol. 13:545586 (1995). Current studies of adoptive transfer in patients with HIV, CMV, and melanoma involve the infusion of T cells that have been stimulated, cloned and expanded for many weeks in vitro on autologous dendritic cells (DC), virally infected B cells, and/or allogeneic feeder cells (Riddell et al., Science 257:238-241 (1992); Yee et al., J. Exp. Med. 192:1637-1644 (2000); Brodie et al.,Nat. Med. 5:34-41 (1999); Riddell et al., Hum. Gene Ther. 3:319-338 (1992), Riddell et al., J. Immunol. Methods 128:189-201 (1990)). However, adoptive T cell immunotherapy clinical trials commonly use billions of cells (Riddell et al., 1995). In order to produce these quantities of cells, 1000-4000 fold expansion of T cells in vitro (10-12 population doublings) is usually required. Furthermore, for optimal engraftment potential and possible therapeutic benefit, it is important to ensure that the T cells, after in vitro expansion, are functional, and not senescent, at the time of re-infusion.
T cell activation is initiated by the engagement of the T cell receptor/CD3 complex (TCR/CD3) by a peptide-antigen bound to a major histocompatibility complex (MHC) molecule on the surface of an antigen-presenting cell (APC) (Schwartz, Science 248:1349 (1990)). While this is the primary signal in T cell activation, other receptor-ligand interactions between APCs and T cells are required for complete activation. For example, TCR stimulation in the absence of other molecular interactions can result in an inability of the T cells to respond to full activation signals upon re-stimulation (Schwartz, 1990; Harding, et al., Nature 356:607 (1992)). In the alternative, T cells die by programmed cell death (apoptosis) when activated by TCR engagement alone (Webb et al., Cell 63:1249 (1990); Kawabe et al., Nature 349:245 (1991); Kabelitz et al., Int. Immunol. 4:1381 (1992); Groux et al., Eur. J. Immunol. 23:1623 (1993)).
Multiple receptor-ligand interactions take place between the T cell and the APC, many of which are adhesive in nature, reinforcing the contact between the two cells (Springer et al., Ann. Rev. Immunol. 5:223 (1987)), while other interactions transduce additional activation signals to the T cell (Bierer e al., Adv. Cancer Res. 56:49 (1991)). CD28, a surface glycoprotein present on 80% of peripheral T cells in humans, is present on both resting and activated T cells. CD28 binds to B7-1 (CD80) or B7-2 (CD86) and is the most potent of the known co-stimulatory molecules (June et al., Immunol. Today 15:321 (1994); Linsley et al., Ann. Rev. Immunol. 11:191 (1993)). Moreover, CD28 ligation on T cells in conjunction with TCR engagement induces the production of IL-2 molecules (June et al., 1994; Jenkins et al., 1993; Schwartz, 1992), which are critical for continued proliferation.
Co-stimulation of T cells has been shown to affect multiple aspects of T cell activation (June et al., 1994). It lowers the concentration of anti-CD3 required to induce a proliferative response in culture (Gimmi et al., Proc. Natl. Acad. Sci. USA 88:6575 (1991)). CD28 costimulation also markedly enhances the production of lymphokines by helper T cells through transcriptional and post-transcriptional regulation of gene expression (Lindsten et al., Science 244:339 (1989); Fraser et al., Science 251:313 (1991)), and can activate the cytolytic potential of cytotoxic T cells. Inhibition of CD28 co-stimulation in vivo can block xenograft rejection, and allograft rejection is significantly delayed (Lenschow et al., Science 257:789 (1992); Turka et al., Proc. Natl. Acad. Sci. USA 89:11102 (1992)).
However, methods of cloning and expanding T cells for adoptive immunotherapy have proven to have certain drawbacks. The standard culture of pure CD8+ cells is limited by apoptosis, and obtaining a sufficient number of cells to be useful has been particularly difficult. Current cell culture techniques require several months to produce sufficient numbers of cells from a single clone (Riddell et al., 1992; Heslop et al., Nat. Med. 2:551-555 (1996)), which is a problematic limiting factor in the setting of malignancy. Indeed, it is possible that the T cells that are currently infused into patients, may have a limited replicative capacity, and therefore, could not stably engraft to provide long-term protection from disease.
While previous investigators have noted long term qualitative persistence of CTLs in human adoptive transfer protocols, the quantitative level of sustained engraftment has been low (Rosenberg et al., N. Engl. J. Med. 323:570-578 (1990); Dudley et al., J. Immunother. 24:363-373 (2001); Yee et al., Curr. Opin. Immunol. 13:141-146 (2001); Rooney et al., Blood 92:1549-1555 (1998)). Therefore, the present invention offers therapeutic implications because there remains an unmet need for sustained high-level engraftment of human CTLs.
The inventors have previously shown that magnetic beads coated with anti-CD3 and anti-CD28 antibodies can be used as artificial antigen presenting cells (APCs) to support the long-term growth of CD4+ T cells (U.S. Pat. No., 6,352,694; Levine et al., J. Immunol. 159:5921-5930 (1997); Latouche, et al., Nat. Biotechnol. 18:405-409 (2000)). However, it was subsequently shown by the inventors and others that beads or plates coated with anti-CD3 and anti-CD28 antibodies cannot support long-term growth of purified CD8+ T cells (Deeths et al., J. Immunol. 163:102-110 (1999); Laux et al., Clin. Immunol. 96:187-197 (2000)). The CD8+ T cells stimulated with anti-CD3/CD28 initially produce IL-2, but unlike CD4+ T cells, become unresponsive when re-stimulated in vitro with anti-CD3 and anti-CD28. Moreover, this limitation cannot be overcome by the addition of IL-2 to the culture medium (Deeths et al., Eur. J. Immunol. 27:598-608 (1997)), and Bcl-xL induction alone, without complete IL-2 receptor signaling, is not sufficient to mediate cell survival or growth (Lord et al., J. Immunol. 161:4627-4633 (1998); Dahl et al., J. Exp. Med. 191, 2031-2038 (2000)). Additional limitations of using the bead-based system on a wide-scale basis include: the high cost of the beads, the labor intensive process involved in removing the beads from the culture before infusion, and the inability of the beads to expand T cells (Deeths et al., 1999; Laux et al., 2000), plus the bead based system is restricted by a need for GM quality control approval before the start of each application
The TNF receptor family member 4-1BB (CD137) was initially identified by receptor screens of activated lymphocytes (Pollok et al., J. Immunol. 150:771-781 (1993)). The 4-1BB ligand is expressed by activated B cells, dendritic cells, and monocytes/macrophages, all of which can act as APCs (Goodwin et al., Eur. J. Immunol. 23:2631-2641 (1993)). Previous studies have shown that stimulation of 4-1BB on CD8+ T cells prolongs survival of CTLs in vivo and amplifies CD8+-dependent immune responses in mice (Shuford et al., J. Exp. Med. 186:47-55 (1997)). Moreover, as a co-stimulatory molecule in the activation of T cells, 4-1BB signaling is independent from, albeit weaker than, CD28 signaling (Deeths et al., 1997; Hurtado et al., J. Immunol. 158:2600-2609 (1997); Hurtado et al., J. Immunol. 155, 3360-3367 (1995); Saoulli et al.J. Exp. Med. 187:1849-1862 (1998)). Consistent with these data, co-stimulation of 4-1BB has been shown to have anti-viral and anti-tumor effects (Tan et al., J. Immunol. 164:2320-2325 (2000); Melero et al., Nat. Med. 3:682-685 (1997); Melero et al., Eur. J. Immunol. 28, 1116-1121 (1998); DeBenedette et al., J. Immunol. 158:551-559 (1997); Guinn et al., J. Immunol. 162:5003-5010 (1999)).
Progress in designing more efficient T cell expansion systems will be a direct result of an improved understanding of T cell activation. Thus, there has been a long-felt need to find a cell based system that can promote long-term growth of activated CTL cells in a more amenable and effective manner than by means of the bead-based system. A cell based artificial APC would be more amenable to test the influence of cytokines and other co-stimulatory molecules than has been previously possible using microspheric artificial APCs. Such a system would also permit testing of the ability of natural ligands, rather than antibodies, to stimulate T cells.